Process for the manufacture of 5&#39;-purine nucleotide by the fermentation method



United States Patent 3,268,415 PROCESS FOR THE MANUFACTURE OF PURINE NUCLEOTIDE BY THE FERMEN- TATION METHOD Shukuo Kinoshita and Takashi Nara, Tokyo, and Masanaru Misawa, Kawasaki-shi, Japan, assignors to Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, a corporation of Ja an N5 Drawing. Filed Mar. 23, 1964, Ser. No. 354,095 Claims priority, application Japan, Apr. 10, 1963, 38/ 17,615; Sept. 10, 1963, 38/ 47,841 Claims. (Cl. 195-28) This invention relates to an industrial process for the manufacture of 5-purine nucleotides such as 5-inosinic acid (hereinafter referred to as 5-IMP), 5-gu anylic acid (hereinafter referred to as 5-G-MP), 5'-adenylic acid (hereinafter referred to as 5'-A=MP) and 5'-xanthylic acid (hereinafter referred to as 5-XM P) by a fermen-tative method. This invention is characterized especially by cultivating Brevibacterium ammoniagenes, an adenine-requiring mutant, or a guanine-requiring mutant thereof, in a culture medium containing pantothenic acid and thiamine, their related substancesor a natural substance containing the same.

From the results of our study using a chemically defined medium, it has been found that pantothenic acid and thiamine (or their related substances) present in the culture medium play very important roles in fermentations wherein adenine-requiring mutant or guanine-requiring mutant of Brevibacterium amm'oniagens is used. Also they play important roles in fermentations wherein Brevibacterium ammoniagenes ATCC No. 6871 and 6872 the parent strains of said mutantsare used in a culture medium containing purine base, nucleoside or its related substances.

As microorganism useful in the present invention, there are mentioned Brevibacterium ammonz'agenes ATCC .N0. 6781 or 6782 for use in those fermentations in which a culture medium containing purine bases, purine nucleoside or a related substance is employed. There .are mentioned adenine-requiring or guanine-requiring mutants derived from above said strains in those direct fermentations in which the culture medium contains pantothenic acid and thiamine, their related substances, or a natural substance containing the same.

Since two parent strains, ATCC-No. 6871 and ATCC No. 6872, require biotin as growth factor, the mutants derived from these strains naturally also require biotin for growth.

It is necessary to add a small amount of adenine to adenine-requiring mutant and a small amount of guanine to qua-nine-requiring mutant. The amount to be added varies with the strains and the culturing conditions, normally about 1200 ,ug./ml. as the respective bases.

At the optimum amounts of addition of each base, 5- I MP is accumulated in case of adenin-requiring mutant and 5-XMP in the case of guanine-requiring mutant.

In the fermentation employing parent strains (ATCC No. 6871 and 6872) purine base and/or its nucleoside must be added as precurser in an amount more than 0.5 mg./ml. When adding hypoxanthine and inosine, 5- IMP is accumulated, 5'-GMP is accumulated with the addition of guanine and guanosine, and 5'-AMP is ac cumulated with the addition of adenine and adenosine. Natural materials containing their precursors can also be employed. For example, the addition of fermentation broth obtained from a culture of microorganisms having hypoxanthine producing ability as hypoxanthine source is possible.

For seed medium and fermentation medium a suitable amount of pantothenic acid, related compound, or natural Patented August 23, 1966 substance containing the same is added to the basal medium. The amounts used will vary depending upon the specific strain utilized as Well as the culture medium. In general the amount added is between about 0.5 to about 50 micrograms/milliliter g/m1.) based on the pantothenic acid or related compound. The amount of thiamine present in the medium will likewise vary as the strain and the medium change. It is preferable, however, that in general the medium contain from about 0.1 ,ug./ ml. to about 10 ,wgJml. based on the thiamine or related substance.

These substances are added to the basal medium containing a carbon source, an inorganic nitrogen source, in organic salts, an amino acid source, and other compounds which are necessary for the growth of the microorganism which is used. Among culture constituents, phosphates and magnesium salts are important, and in order to obtain desirable yields, it is necessary to add both of them to eifect extremely higher concentration.

The most desirable addition is 0.6-1.5% of K HPO 06-15% of KH PO, and 06-15% of MgSO -7H O, and addition should be effected to obtain nearly equal concentration of these three salts. A natural culture medium which contains pantothenic acid and thiamine or their related compounds is well suited for use in the present invention.

For the strains of Brevibacterium ammoniagenes which are used in the present invention, salts of pantothenic acid such as calcium pantothenate, p-alanine, pantethine (or pantet-hene), coenzyme A and the like are intended as pantothenic acid related substance and may be employed instead of pantothenic acid.

As natural substances containing pantothenic acid which are useful in the instant invention there may be mentioned meat extract, liver extract, rice bran, molasses, fish solubles, and the like.

As natural substances containing thiamine which may be mentioned are those such as rice husk and bran, yeast extract, liver extract, and the like.

As substances which are related to thiamine and which are useful in the instant invention there may be mentioned salts of thiamine such as thiamine hydrochloride, thiamine mononitrate, and the like as well .as other related compounds.

Thus the invention is distinguished especially by incorporating a compound of the pantothenic acid series and a compound of the thiamine series in a culture medium used in the fermentation of either (1) Brevibacterium ammtmiagene's ATCC No. 6871 or ATCC No. 6872 or (2) an adenine-requiring mutant or guanine-requiring mutant of said Brevibacterium amimoniagenes to form 5- purine nucleotides such as 5-IMP, S'-GMP, S AMP, 5'- XMP, etc. When the fermentation is carried out utilizing the Brevibacte'rium ammoniagenes ATCC No. 6871 or 6872 it is conducted in the presence of purine base or its nucleoside in the medium. In the case of the fermentation of the adenine or guanine-requiring mutants of the Brevibacterium am-moniagenes the cultivation is carried out in the medium containing a small amount of adenine (for adenine-requiring mutants) or guanine (for guaninerequiring mutants) to form the said 5'-purine nucleotide.

The method of fermentation is substantially the same as other methods. The temperature is 25 -38 C., the optimum being about 30 C. Normally, shaking is desirable for flask and submerged culturing is desirable for tank and jar fermentor. Culturing time is 24-168 hours,

amount of 5'-purine nucleotide reaches the maximum with ous modifications may be performed without departing either from the spirit or scope of the present invention. The percent of medium composition described in the following examples shows that of each component dissolved in water, based on total volume.

EXAMPLE 1 Brew'bacrerium ammoniagenes ATCC No. 6872 is cultivated for 24 hrs. after autoclaving (at 120 C. for 15 min.) the seed culture medium containing 2% glucose, 1.0% casamino acids (free from vitamin), 0.1% K I-IP 0.03% MgSO -7H O, 0.3% NaOl, 0.01% FeSO -7H O and 30 gJ-l. biotin (pH 7.3), and inoculated in a proportion of 10% (volume) to the fermentation medium. Each 30 ml. of the media is poured into a 250 ml. Erlenmeyer flask and used after autoclaving (at 120 C. for min). The fermentation. medium of the following composition is used and the cultivation is performed at 30 C. with stirring.

The composition of fermentation medium: 10% glucose, 1% K HPO 1% KH PO 1% MgSO -7H O, 0.01% CaCl '2H O, 30 ,ug./l. biotin, 3 mg/ml ]1y poxanthine, Ca-pantothenate in various concentrations. The pH value is adjusted with 5 N-NaOH to 8.0 before autoclaving (at 120 C. for 15 min). Following sterilization, 0.6% sterilized urea and thiamine-H01 in various concentrations are added to said medium. The amount of 5'-inosinic acid (Na-salt) accumulated in the cultivating liquor after 96 hrs. is shown in the following Table I.

Table 1 Calcium panto- Thiamine HCl 5-inosi.uic acid thenate added added g/ml.) (Na-salt) formed (ea/ e/ 0 0 trace 5 0 trace 0 1 trace EXAMPLE 2 The cultivation is performed, using the same strain and under cultivation conditions similar to those described in Example 1 with the addition of fl-alanine in various concentrations instead of calcium pantothenate. The amount of 5'-inosinic acid (Na-salt) accumulated after 120 hrs. is shown in Table 11 below.

The same strain as used in Example 1 and the same seed medium as used in Example 1 is used except for adding 1.5% .pept-one instead of 1.0% casamino acids. The fermentation medium used is one to which suit-able amounts of B-alanine, rpan tet'hine, coenzyme A and the like and yeast extract, meat extract, liver extract, fish solubles of natural material containing these compounds are added in place of calcium pantothenate, and furthermore to which 1 ng./ ml. of thiamine is added. Other cultivation conditions are similar to that of Example 1. The

amount of 5'-inosinic acid (Na-salt) accumulated in the fermentation medium after cultivating for hrs. is shown in Table III below.

EXAMPLE 4 Brevibacterium ammoniagenes ATCC No. 6871 is used. The same seed and fermentation medium as used in Example 3 are used and the strain is cultivated adding pantothenic acid (or fi-alanine) and thiamine in various concentrations. Other fermentation procedures are similar to that of Example 1. The amount of 5-inosinic acid (Na-salt) accumulated after cultivating for 96 hrs. is shown as in Table IV below.

Table IV Thiamine 5-inosi.uie acid Compound of pantcthenic acid series added (N a-salt) added g/ml.) accumulated Not added Not added--- Trace. Calcium pantothenate, 10 gJml Not added- Trace. Not added... 2 Trace. Calcium pantothenate, 10 gJml 2 8.9. fl-Alinine, 10 u Iml 2 9.2.

EXAMPLE 5 Br. ammoniagenes No. 7208, ATCC No. 15187 (adenine-requiring mutant derived from ATCC No. 6872) is used. The medium obtained by adding 50 ,ugJml. adenine to the medium described in Example 1 is used as seed medium and the medium obtained by adding 50 ,ug/ml. adenine to the medium described in Example 1 is used as fermentation medium. The strain is cultivated adding suitable amount'of calcium pantothenate (or fi-alanine) and thiamine to the fermentation medium. Other cultivation conditions are similar to that of Example 1. The amount of 5'-inosinic acid (Na-salt) accumulated after cultivating for 120 hrs. is shown in Table V below.

Br. ammoniagenes No. 7244, ATCC No. 15188 (adenineand xanthine-requiring mutant derived from ATCC N0. 6872) is used. The seed medium and fermentation medium described in Example 5 are used with the addition to each of 40 ,ugJrnl. xanthine. The medium without the addition of and the medium with the addition of 10 pg/ml. calcium pantothenate and 1 ,ugJml. thiamine are cultivated respectively. Other cultivation conditions are similar to that of Exampl 1. The amount of 5-inosinic acid (Na-salt) accumulated after cultivating for 120 EXAMPLE 7 Br. ammonz'agenes No. 7309, ATCC No. 16 312 (adenine-requiring mutant derived from ATCC No. 6872) is used and the seed medium and fermentation medium described in Example 5 in which the amount of adenine added is 5 ,ugjml. are used. The medium without the addition of and the medium with the addition of ,ug./ ml. calcium pantothenate and 1 ug./ ml. thiamine are cultivated respectively. Other cultivation conditions are similar to that of Example 1. The amount of 5-inosinic acid accumulated after cultivating for 96 hrs. is trace in the case where no vitamin is added and 6.3 mg./ml. in the case where both vitamins are added.

EXAMPLE 8 The same strain and culture medium as described in Example 7 are used and the fermentation medium to which is added fiaalani-ne, Ipantethin'e, coenzyme A and natural substance containing ,B-ala-nine, paute'thine and coenzyme A as a substitute tEor calcium pantothenate are used. Other cultivation conditions are similar to that of Example 1. The amount of 5-inosinic acid (Na-salt) accumulated after cultivating for 120 hrs. is shown in Table VI below.

EXAMPLE 9 Br. ammoniagenes 7320, ATCC No. 190 (adeninerequiring mutant derived from ATCC No. 687 1) is used, and other cultivation conditions are similar to that of Example 5. Arfter cultivating for 9 6 hrs., 4.2 rug/ml. of 5'-inosinic acid (Na-salt) is accumulated in the medium 'which is obtained by adding 5 rig/ml. S-alanine and 1 ,ugjml. thiamine \to the fermentation meditun. On the other hand, 5'-inosinic acid is only slightly accumulated in the medium tree from both vitamins.

EXAMPLE 10 The same strain and seed medium as in Example 1 are used, the medium which is obtained by adding 0.3% peptone to the fermentation medium described in Example 1 without hypoxanthine is used. Pantothenic acid or its related compound and thiamine are added thereto in various concentrations. The addition of guanine during the growth is penformed in such manner that 2 mg./ml. guanine is obtained after cultivating for 72 1118. Other cultivation conditions are similar to that of Example 1. The amount of 5-guanylic acid (Na-salt) accumulated in the cultivating liquor after cultivating for 9 6 hrs. is shown in Table VII below.

6 EXAMPLE 11 The same strain and seed medium as described in Example 1 are used and the fermentation medium similar to that of Example 10, to which S g/ml. pantothenic acid and 1 ug./ ml. thiamine-HCl are added, is use-d. The addition is penformed in such manner that 2.5 mg./-ml. adenine is obtained after cultivating for 48 hrs. Other cultivation conditions are similar to that of Example 1. 6.5 mg./ml. of 5'-adenylic acid is accumulated in the cultivating-liquor after cultivating for 96 hrs. 'When either-pantothenic acid or thiamine is omitted, 5'-adenylic acid is only slightly accumulated.

EXAlMP LE 12 Br. ammoniagenes ATCC N0. 6871 is used. The seed medium and fermentation medium described in Example 1 0 are used and the component added in such manner that 2.0 mg./ml. guanine or adenine is obtained after cultivating for 48 hrs. Other cultivation conditions are similar to that 01f Example '1. Thus, in the cultivating liquor after cultivating for 96 hrs, 4.8 mg./ml. of 5-guanylic acid (Na-salt) is accumulated in the.case where guanine is added and 5.1 mg./rnl. orf 5-adenylic acid is accumulated in the case where adenine is added. When either pantothenic acid or thiamine is omitted, 5'-guanylic or 5-adenylic acid is only slightly accumulated.

EXAMPLE 13 Table VIII Pantothenic acid related compound Thiamine acid (Na-salt) added (#gJml.) added g/m1.) accumulated (mg.rul.)

Calcium pantothenate, 5 0 trace Not added 1 trace Calcium pantothcnate, 5.. 1 7. 2 Pantethine, 4 1 7. 5 Coenzyrne A, 25 1 7. 6

What is claimed is:

1. A process for the manufacture of 5'-purine nucleotide selected from the group consisting of 5'-inosinic acid, 5'-guanylic acid, 5-adenylic acid and 5'-xanthylic acid, wherein one of the microorganisms Brevibacterium nmmoniagenes ATCC No. 6871, Breyibacterium ammoniagenes ATCC No. 6872 and mutants thereof, is cultivated in a culture medium containing (1) from about 0.5 to about 50 micrograms per milliliter of a member selected from the group consisting of pantothenic acid, pantothenic acid-yielding substance, ii-alanine, pantethene and coenzyme A and (2) from about 0.1 to about 10 micrograms per milliliter of a member selected from the group consisting of thiamine, thiamine hydrohalide and thiamine-yielding substance, said culture medium also containing biotin and a purine base or a nucleoside thereof, until essentially maximum accumulation of objective 5'-nucleotide has been achieved, and then recovering the thusaccumulated 5-nucleotide from the culture medium.

2. A process according to claim 1, wherein the culture medium contains 0.61.5% of K HPO 06-15% of KH PO and 0.6-1.5 of MgSO -7H O.

3. A process according to claim 2 wherein the culture medium also contains a growth-promoting amount of biotin and an amount of a purine base selected from the group consisting of adenine, xanthine, guanine and mixture thereof to satisfy the purine base requirement of the microorganism.

4. A process as claimed in claim 1 wherein the microorganism is Brevibacterium ammonz'agenes ATCC No. 6871.

5. A process as claimed in claim 1 wherein the microorganism is Brevibacterium ammoniagenes ATCC No.

6. A process as claimed in claim 3 wherein the microorganism is Brevz'bacterium ammoniagenes No. 7208 (ATCC No. 15187).

7. A process as claimed in claim 3 wherein the microorganism is Brevibacterium ammoniagenes No. 7244 (ATCC No. 15188).

8. A process as claimed in claim 3 wherein the microorganism is Brevibacterium ammoniagenes No. 7309 (ATCC No. 15312).

9. A process as claimed in claim 3 wherein the microorganism is Brevibacterium ammoniagenes No. 7320 (ATCC No. 15190).

10. A process as claimed in claim 3 wherein the micro organism is Brevibacterium ammoniagenes No. 62221 (ATCC No. 15138).

References Cited by the Examiner UNITED STATES PATENTS 3,118,820 1/1964 Uchida et a1. 195-28 3,135,666 6/1964 Hara et a1 195-28 3,152,966 10/1964 Kinoshita et a1 19528 FOREIGN PATENTS 1,316,247 12/ 1962 France.

A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Examiner. 

1. A PROCESS FOR THE MANUFACTURE OF 5''-PURINE NUCLEOTIDE SELECTED FROM THE GROUP CONSISTING OF 5''-INOSINIC ACID, 5''GUANYLIC ACID, 5''-ADENYLIC ACID AND 5''-XANTHYLIC ACID, WHEREIN ONE OF THE MICROORGANISMS BREVIBACTERUM AMMONIAGENES ATCC NO. 6871, BREVIBACTERIUM AMMONIAGENES ATCC NO. 6872 AND MUTANTS THEREOF, IS CULTIVATED IN A CULTURE MEDIUM CONTAINING (1) FROM ABOUT 0.5 TO ABOUT 50 MICROGRAMS PER MILLILIER OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF PANTOTHENIC ACID, PANTOTHENIC ACID-YIELDNG SUBSTANCE, B-ALANINE, PANTETHENE AND COENZYME A AND (2) FROM ABOUT 0.1 TO ABOUT 10 MICROGRAMS PER MILLILITER OF A MEMBER SELECTED FROM THE GROUP CONSISTING OF THIAMINE, THIAMINE HYDROHALIDE AND THIAMINE-YIELDING SUBSTANCE, SAID CULTURE MEDIUM ALSO CONTAAINING BIOTIN AND A PURINE BASE OR A NUCLEOSIDE THEREOF, UNTIL ESSENTIALLY MAXIMUM ACCUMULATION OF OBNJECTIVE 5''-NUCLEOTIDE HAS BEEN ACHIEVED, AND THEN RECOVERING THE THUS-ACCUMULATED 5-NUCLEOTIDE FROM THE CULTURE MEDIUM. 